Retinitis pigmentosa (RP) is a group of human hereditary retinal degenerations characterized by night blindness and loss of peripheral vision. RP sometimes progresses to total blindness. Cardinal clinical features of RP include retinal pigmentary disturbances, attenuation of the retinal vasculature, a waxy pale appearance of the optic nerve head, and abnormalities of retinal function. RP is the most common mendelian degenerative retinal disorder, affecting 1.5 million individuals worldwide (Kumar Singh, R., Farrar, G. J., Mansergh, F., Kenna, P., Bhattacharya, S., Gal, A. and Humphries, P. (1993) Hum. Mol. Genet. 2, 875-878). Among Caucasians in the United States, when not associated with other abnormalities, RP is inherited most frequently as an autosomal recessive (84% of cases), next as an autosomal dominant (10%), and least frequently as an X-linked recessive disorder (6%) (Boughman, J. A., Conneally, P. M. and Nance, W. E. (1980) Am. J Hum. Genet. 32, 223-235).
Significant nonallelic heterogeneity has been found in autosomal dominant RP (adRP) and in X-linked RP. For adRP, mutations in the rhodopsin gene on 3q (Sung, c. H., Davenport, C. M., Hennessey, J. C., Maumenee, I. H., Jacobson, S. G., Heckenlively, J. R., Nowakowski, R., Fishmanm, G., Gouras, P. and Nathan, J. (1991) Proc. Natl. Acad. Sci. USA 88, 6481-6485; Ingleheam, C. F., Keen, T. J., Bashir, R., Jay, M., Fitzke, F., Bird, A. C., Crombie, A. and Bhattacharya, S. (1992) Hum. Mol. Genet. 1, 41-45; Humphries, P., Kenna, P. and Farrar, G. J. (1992) Science 256, 804-808), and the peripherin gene on 6p have been found (Farrar, G. J., Kenna, P., Jordan, S. A., Kumar Singh, R., Humphries, M. M., Sharp, E. M., Sheils, d. M. and Humphries, P. (1991) Nature 354, 478-480; Kajiwara, K., Hah, L. B., Mukai, S., Travis, G. H., Berson, E. L. and Dryja, T. P. (1991) Nature 354, 480-483; Farrar, G. J., Kenna, P., Jordan, S. A., Kumar Singh, R., Humphries, M. M., Sharp, E. M., Sheils, D. and Humphries, P. (1993) Genomics 15, 466). In addition, genetic linkage of adRP is observed to loci on chromosomes 8 (Blanton, S. H., Heckenlively, J. R., Cottingham, A. W., Friedman, J., Sadler, L. A., Wagner, M., Friedman, L. H. and Daiger, S. P. (1991) Genomics 11, 857-869), 7p (Inglehearn, C. F., Carter, S. A., Keen, T. J., Lindsey, J., Stephenson, A. M., Bashir, R., al Maghtheh, M., Moore, A. T., Jay, M., Bird, A. C. and Bhattacharya, S. S. (1993) Nature Genet. 4, 51-53), 7q (Jordan, S. A., Farrar, G. J., Kenna, P., Humphries, M. M., Sheils, D. M., Kumar Singh, R., Sharp, E. M., Soriano, N., Ayuso, C., Benitez, J. and et al, (1993) Nature Genet. 4, 130-134), and 19 (Al-Maghtheh, M., Inglehearn, C. F., Keen, T. J., Evans, K., Moore, A. T., Jay, M., Bird, A. C. and Bhattacharya, S. S. (1994) Hum. Mol. Genet. 3, 351-354). For the X-linked form of the disorder, two loci have been implicated (Ott, J., Bhattacharya, S., Chen, J. D., Denton, M. J., Donald, J., Dubay, C., Farrar, G. J., Fishman, G. A., Frey, D., Gal, A., Humphries, P., Jay, B., Jay, M., Litt, M., Machler, M., Musarella, M., Neugebauer, M., Nussbaum, R. L., Terwilliger, J. D., Weleber, R. G., Wirth, B., Wong, F., Worton, R. G. and Wright, A. F. (1990) Proc. Natl. Acad. Sci. USA 87, 701-704). Genetic linkage has not been reported for autosomal recessive RP (arRP), but mutations that cosegregate with arRP have been found in the xcex2-subunit of rod phosphodiesterase on chromosome 4p (McLaughlin, M. E., Sandberg, M. A., Berson, E. L. and Dryja, T. P. (1993) Nature Genet. 4, 130-134), rhodopsin (Rosenfeld, P. J., Cowley, G. S., McGee, T. L., Sandberg, M. A., Berson, E. L. and Dryja, T. P. (1992) Nature Genet. 1, 209-213), and possibly the rod cGMP-gated channel gene (McGee, T. L., Lin, D., Berson, E. L. and Dryja, T. P. (1994) Invest. Ophthalmol. Vis. Sci. (Suppl.) 35, 1716 (Abstract)). The regions around rhodopsin and peripherin have been excluded as a cause of arRP in a large Dutch pedigree (Bleeker Wagemakers, L. M., Gal, A., Kumar Singh, R., Ingeborgh van den Born, L., Li, Y., Schwinger, E., Sandkuijl, L. A., Bergen, A. A., Kenna, P., Humphries, P. and Farrar, G. J. (1992) Genomics 14, 811-812), suggesting that the recessive form of the disorder is also genetically heterogeneous.
A locus for autosomal recessive retinitis pigmentosa has been mapped on chromosome 6p by linkage analysis (Knowles, J. A., et. al., (1994) Hum. Mol. Genet. 3:1401-1403). This locus is approximately 20 centimorgans telomeric from the previously described adRP disease gene, peripherin, and thus represents a novel disease locus. The approximate 95% confidence interval for arRP spanning the D6S291 locus is 8.5% centimorgans. This interval of chromosome 6p contains the HLA complex. HLA associations have been found in several human uveitic diseases (Nussenblatt, R. B. et al., (1989) Uveitis: Fundamentals and Clinical Practice: Year Book Medical Publishers), and the 1-A subregion of the H-2 complex in a mouse model of autoimmune uveoretinitis (Caspi, R. R., et. al., (1992), Curr. Eye Res. 11 (Suppl.) 81-86; Caspi, R. R., et, al., (1992) J. Immunol., 148:2384-2389).
The present invention is based on the discovery of novel molecules, referred to herein as rp nucleic acid and polypeptide molecules. Exemplary rp molecules, the first containing a Bac clone genomic DNA and the second containing the cDNA shown in FIGS. 1A-1B have been deposited with the American Type Culture Collection (ATCC) and have been assigned ATCC designation numbers: 98144 (deposited Aug. 22, 1996) and 98147 (deposited Aug. 23, 1996), respectively.
The human rp gene (FIG. 1), which is approximately 1050 nucleotides in length, is predominantly expressed in the retina, consistent with the phenotypic abnormalities seen in Retinitis Pigmentosa patients. In addition, the gene was found to map very close (2.8 cR or about 1 Mb) to marker D6S291 on chromosome 6. This marker is the most tightly linked marker to the RP14 retinitis pigmentosa locus. The human rp gene encodes a 349 amino acid protein, that weighs approximately 38880 daltons.
The human rp gene is 56.4% identical to the human tub gene; and the hrp protein is 46% identical to htub. Although the N-terminus of the two gene products are dissimilar, the most conserved region of the two gene products are 66.4% identical. Accordingly and as discussed further herein, the tub and rp genes may perform similar molecular functions in different biochemical pathways or may even function in the same biochemical pathway(s).
In one aspect, the invention features isolated vertebrate rp nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional rp polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of the human rp polypeptide). In one embodiment, the nucleic acid molecules hybridize to the rp gene contained in ATCC Designation Nos. 98144 or 98147 or to the complement of the rp gene contained in ATCC Designation Nos. 98144 or 98147. In another embodiment, the nucleic acids of the present invention can hybridize to a vertebrate rp gene or to the complement of a vertebrate rp gene. In a further embodiment, the claimed nucleic acid hybridizes with the coding sequence designated in at least one of SEQ ID Nos: 1 or 3 or to the complement to the coding sequence designated in at least one of SEQ ID Nos: 1 or 3. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is a rp nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 95% homologous in sequence to the nucleic acids shown as SEQ ID Nos: 1 or 3 or to the complement of the nucleic acids shown as SEQ ID Nos: 1 or 3. In another embodiment, the rp nucleic acid molecule encodes a polypeptide that is at least 90% and more preferably at least 94% similar in sequence to the polypeptide shown in SEQ ID No: 2. In a further embodiment, the nucleic acid molecule is a rp nucleic acid that is at least 70%, preferably 80%, more preferably 85% and even more preferably at least 90% or 95% similar in sequence to the rp gene contained in ATCC Designation Nos. 98144 or 98147.
The invention also provides probes and primers comprising substantially purified, oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least 6 consecutive nucleotides of the sequences set forth as SEQ ID Nos: 1 or 3 or complements of the sequences set forth as SEQ ID Nos: 1 or 3, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject rp nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence, which regulatory sequence is operably linked to the rp gene sequence. Such regulatory sequences in conjunction with a rp nucleic acid molecule can be useful vectors for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing rp proteins by employing said expression vectors.
In another aspect, the invention features isolated rp polypeptides, preferably substantially pure preparations e.g. of plasma purified or recombinantly produced rp polypeptides. In one embodiment, the polypeptide is identical to or similar to a rp protein represented in SEQ ID No: 2. Related members of the vertebrate and particularly the mammalian rp family are also within the scope of the invention. Preferably, a rp polypeptide has an amino acid sequence at least 66.5% homologous and preferably at least 70, 80, 85, 90 or 95% homologous to the polypeptide represented in SEQ ID No: 2. In a preferred embodiment, the rp polypeptide that is encoded by a nucleic acid which hybridizes with a nucleic acid sequence represented in one of SEQ ID Nos: 1 or 3 or with the gene or gene fragment contained in ATCC Designation Nos. 98144 or 98147. The subject rp proteins also include modified protein, which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The rp polypeptide can comprise a full length protein, such as represented in SEQ ID No: 2, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250,275, 300, 325, 330, 335 or 340 amino acids in length. A particularly preferred rp polypeptide is comprise of 349 amino acids and has a molecular weight of about 38880 daltons.
Another aspect of the invention features chimeric molecules (e.g. fusion proteins) comprised of a rp protein. For instance, the rp protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the rp polypeptide (e.g. the second polypeptide portion is glutathione-S-transferase, an enzymatic activity such as alkaline phosphatase or an epitope tag).
Yet another aspect of the present invention concerns an immunogen comprising a rp polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a rp polypeptide; e.g. a humoral response, an antibody response and/or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the protein represented in SEQ ID No: 2.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the rp protein. In preferred embodiments the antibody specifically binds to at least one epitope represented in SEQ ID No: 2.
The invention also features transgenic non-human animals which include (and preferably express) a heterologous form of a rp gene described herein, or which misexpress an endogenous rp gene (e.g., an animal in which expression of one or more of the subject rp proteins is disrupted). Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed rp alleles or for use in drug screening. Alternatively, such a transgenic animal can be useful for expressing recombinant rp polypeptides.
In yet another aspect, the invention provides assays, e.g., for screening test compounds to identify inhibitors, or alternatively, potentiators, of an interaction between a rp protein and, for example, a virus, an extracellular ligand of the rp protein, or an intracellular protein which binds to the rp protein. An exemplary method includes the steps of (i) combining a rp polypeptide or bioactive fragments thereof, a rp target molecule (such as a rp ligand or a rp substrate), and a test compound, e.g., under conditions wherein, but for the test compound, the rp protein and target molecule are able to interact; and (ii) detecting the formation of a complex which includes the rp protein and the target polypeptide either by directly quantitating the complex, by measuring inductive effects of the rp protein, or, in the instance of a substrate, measuring the conversion to product. A statistically significant change, such as a decrease, in the interaction of the rp and target molecule in the presence of a test compound (relative to what is detected in the absence of the test compound) is indicative of a modulation (e.g., inhibition or potentiation of the interaction between the rp protein and the target molecule).
Yet another aspect of the present invention concerns a method for modulating the transcription of certain genes in a cell by modulating rp bioactivity, (e.g., by potentiating or disrupting rp bioactivity). In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a rp therapeutic so as to alter, relative to the cell in the absence of treatment, the level of transcription of certain genes Accordingly, the method can be carried out with rp therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a rp protein or ligand binding of a rp protein. Other rp therapeutics include antisense constructs for inhibiting expression of rp proteins, and dominant negative mutants of rp proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type rp protein.
A further aspect of the present invention provides a method of determining if a subject is at risk for Rieger Syndrome or other disorder resulting from a mutant rp gene. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a rp protein, e.g.a gene represented in one of SEQ ID Nos: 1 or 3 or a homolog thereof; or (ii) the misexpression of a rp gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a rp gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble rp protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of a rp gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the rp gene; (ii) contacting the probe/primer to an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the rp gene and, optionally, of the flanking nucleic acid sequences. For instance, the primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a rp protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the rp protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.